Fig. 1.

Transfer of [¹³C]cholesterol from [¹³C]cholesterol-loaded macrophages into adjacent SMCs. The SMCs had been grown for 21 d in medium containing [¹⁵N]choline. [¹³C]cholesterol-loaded macrophages were plated onto a ∼90 to 95% confluent monolayer of SMCs and incubated overnight at 37 °C in serum-free medium. NanoSIMS images were recorded from the interior of cells (note the presence of nuclei with negligible amounts of ¹⁵N enrichment). (A) A mosaic of ¹³C/¹²C and ¹⁵N/¹⁴N NanoSIMS images centered on a [¹³C]cholesterol-loaded macrophage. The macrophage is noted with a yellow arrow. The large black holes in the NanoSIMS images represent regions of the silicon wafer that were not covered by cells. (Scale bars: 10 μm.) Ratio scales were multiplied by 10,000. The natural abundance of ¹³C is 1.1%. ¹²C^–, ³²S^–, and ¹²C¹⁴N^– NanoSIMS images of the same field are shown in SI Appendix, Fig. S2. (B) ¹³C/¹²C ratio (mean ± SD) in macrophage (Mac) cytosolic lipid droplets (LD) (n = 23 LDs in five regions of 45 μm × 45 μm), LDs in SMCs immediately adjacent to the macrophage (n = 52 LDs in five regions of 45 μm × 45 μm), and LDs in more distant SMCs (n = 42 LDs in four regions of 45 μm × 45 μm). The cytosolic LDs in SMCs and macrophages were identified in the ³²S^– and ¹²C¹⁴N^– NanoSIMS images (SI Appendix, Fig. S2). (C) High magnification image of the [¹³C]cholesterol-loaded macrophage shown in A. Ratio scales were multiplied by 10,000. The ratio scale of 125–175 shows the transfer of [¹³C]cholesterol from Macs to SMCs; the ratio scale of 110–400 shows [¹³C]cholesterol enrichment in Macs; and the ratio scale of 110–2,000 shows [¹³C]cholesterol enrichment in cytosolic LDs in Macs. Note that the macrophage is relatively enriched in ³²S. Red arrows show a few examples of cytosolic LDs and yellow arrow points to the macrophage. (Scale bar: 10 μm.)